Colorectal cancer (CRC) is the second leading cause of cancer death in the United States, due to the fact that chemoresistance develops in nearly all patients leading to ~50,000 deaths each year. Targeted therapies (such as anti-angiogenesis) for metastatic CRC have a limited impact of patient outcomes, leaving the majority of patients with unresectable metastatic disease dying within 2 years. There is accumulating evidence for the existence of colorectal cancer stem cells (CSC), which mediate the cancer relapse after chemotherapy. CSC phenotype can be influenced by the tumor microenvironment. Thus, modulation of the tumor microenvironment could be a future strategy to reverse the CSC phenotype. This approach requires a better understanding of the microenvironmental factors and mechanisms that regulate the CSC phenotype. Our preliminary studies demonstrate that endothelial cells (EC) secrete soluble factors that enhance the CSC phenotype and chemoresistance of CRC cells. We hypothesize that tumor ECs not only form the microvasculature network providing a conduit for nutrient and oxygen delivery, but also contribute paracrine factors to the tumor microenvironment that mediate the CSC phenotype and chemoresistance. The following specific aims are designed to test this hypothesis. Specific Aim 1: To determine the effect of freshly isolated human ECs on promotion of the CSC phenotype, chemoresistance, and activated pathways in CRC cells in vitro. Specific Aim 2: To identify factors secreted by ECs that mediate the induction of the colorectal CSC phenotype and chemoresistance. Specific Aim 3: To validate the paracrine effect of ECs on promoting the colorectal CSC phenotype in vivo. The overall goal of this proposal is to provide new insights into the role of ECs in the tumor microenvironment. Our proposed study will identify the EC paracrine factors that regulate the CSC phenotype in CRC cells, and this will form the foundation for the development of new therapeutics for metastatic CRC. Blockade or intervention of aberrant EC paracrine signaling will be incorporated into anti-cancer regimens, in addition to anti-angiogenic agents, to improve the outcome of patients with metastatic CRC. PUBLIC HEALTH RELEVANCE: There is accumulating evidence for the existence of a population of colorectal cancer cells within the main tumor mass, that are responsible for the initiation of cancer and resistance to chemotherapy; these cells are termed cancer stem cells. Our laboratory has found that the cells lining blood vessels, endothelial cells, can secrete factors that have the ability to enhance the number of cancer stem cells within the tumor. The ultimate goal of this project is to discover novel therapeutic targets secreted by endothelial cells in order to decrease the percent of cancer stem cells within a tumor and to improve the therapeutic outcome of patients with metastatic colorectal cancer.